tRNA Synthetase Fidelity Mechanisms

描述(由申请人提供)： 氨基酰-tRNA合成酶(AARs)包括一个多达22种酶的家族，这些酶对每个活着的有机体都是必不可少的。每种酶识别一种同源氨基酸，并将其共价连接到正确的tRNA上。“带电的”tRNA然后转移核糖体上的氨基酸，以特定的方式并入不断增长的多肽链。蛋白质合成的保真度完全依赖于AARS对底物的准确识别，AARS可以预防神经疾病。一些AARS已经开发了编辑机制来纠正未被激活的氨基酸。这些编辑的AARs通过两种底物中的任何一种的水解来清除错误的氨基酸--失活的氨基酰基腺苷(将氨基酸“预转移”为tRNA)或错酰化的AA-tRNA(“转移后”)。尽管这些机制中的一种可能占主导地位，但大多数编辑的AARS似乎是通过转移前和转移后编辑的混合来操作的，这在历史上需要复杂的研究来确定它们各自的分子基础。亮氨酰-tRNA合成酶(Leucyl-tRNA Synthetase，LEUR)的新模型已经被开发出来，用于分离转移前编辑活动，以便进行详细的机制研究。此外，转移前编辑可以依赖于tRNA。假设tRNA在合成域和编辑域之间的移位控制着酶在氨酰化、转移后编辑和转移前编辑之间的路径选择。这项建议概述了一个跨学科的研究计划，它结合了X射线结晶学、计算、单分子荧光、生化和分子生物学的方法，以研究tRNA易位和形成稳定的氨基酰化和编辑络合物的酶-tRNA转变的分子基础。它还将描述传输前编辑及其对细胞的生理影响。对编辑机制的详细了解将有助于正在进行的药物研究，这些研究利用AARS，特别是LEURS编辑站点，作为抗生素开发的目标。它还将使AARs能够重新设计，以激活替代氨基酸，并将其整合到定制设计的蛋白质中。这些新的蛋白质可以作为医疗和技术应用中的治疗或工具。

DESCRIPTION (provided by applicant): The aminoacyl-tRNA synthetases (aaRSs) comprise a family of up to twenty two enzymes that are essential to every living organism. Each enzyme recognizes a single cognate amino acid and covalently attaches it to the correct tRNA. The "charged" tRNA then transfers the amino acid at the ribosome for specific incorporation into the growing polypeptide chain. The fidelity of protein synthesis is completely dependent on accurate substrate recognition by the aaRSs, which guards against neurological disease. Some aaRSs have developed editing mechanisms to correct misactivated amino acids. These editing aaRSs clear the wrong amino acid by hydrolysis of either of two substrates-misactivated aminoacyl-adenylates ("pre-transfer" of amino acid to tRNA) or misacylated aa-tRNA ("post-transfer"). Although one of these mechanisms may dominate, most aaRSs that edit appear to operate by a mixture of pre-and post-transfer editing, which has historically complicated investigations to determine their respective molecular basis. New models for leucyl-tRNA synthetase (LeuRS) have been developed to isolate pre-transfer editing activities for detailed mechanistic investigations. In addition, pre-transfer editing can be dependent on tRNA. It is hypothesized that tRNA translocation between the synthetic and editing domains controls the enzyme's pathway choice between aminoacylation, post-transfer editing, and pre-transfer editing. This proposal outlines an interdisciplinary research plan that combines X-ray crystallography, computation, single molecule flourescence, biochemical, and molecular biology approaches to investigate tRNA translocation and the molecular basis of the enzyme- tRNA transition that forms stable aminoacylation and editing complexes. It will also characterize pre-transfer editing and its physiological impact on the cell. A detailed understanding of editing mechanisms will benefit ongoing pharmaceutical research that capitalizes upon aaRSs, especially the LeuRS editing site, as targets for antibiotic development. It will also enable re-engineering of aaRSs to activate alternate amino acids for incorporation into custom-designed proteins. These novel proteins could be used as therapeutics or tools in medicinal and technological applications.